Polysialic acid in alpha2->8 linkage is a component of a neural cell adhesion molecule, N-CAM, and is developmentally regulated. Polymers containing >55 sialyl residues were recently demonstrated on glycoproteins of human neuroblastoma cells, providing a human model system to study the oligosaccharide containing polysialic acid and the expression and regulation of the enzyme, CMP-NeuNAc: poly alpha2->8sialosyl sialyltransferase (polyalpha2->8NeuNActransferase) on a molecular level. The detailed structures of the oligosaccharide residues to which polysialic acid is glycosidically bound will be determine after isolation of glycopeptides enriched in polysialic acid from the human be defined by 500- MHz 'H-NMR spectroscopy in collaboration with J.F.G. Vliegenthart, Utrecht before obtained by lectin affinity, size and charge chromatography. Polyalpha2->8NeuNActransferase will be purified from human neuroblastoma and used to obtain molecular probes for the enzyme. A rapid assay for enzyme activity will be developed, utilizing the information obtained from the NMR spectroscopy of the polysialic acid-containing glycopeptides to define the substrate. Purification of the enzyme after detergent extraction will be by chromatofocusing and substrate affinity chromatography. Probes will be generated from the purified enzyme by two approaches and used to screen genomic or cDNA libraries: 1) synthesis of oligonucleotides which will be deduced from a partial amino acid sequence of the purified enzyme and 2) preparation of antibodies to the enzyme or synthetic peptides deduced from the amino acid sequence information. After characterization of the inserts by nucleotide sequence and by hybrid select translational studies, the inserts coding for the enzyme will be used as probes to examine the molecular mechanisms of the regulation of the enzyme in neuroblastoma. The probes will also be used to define the mechanisms underlying the developmental regulation of the enzyme in embryonic and adult rat brain. These findings will be correlated to the expression of the enzyme in neuroblastoma and, if present, in other neuronal tumors. Neuroblastoma and other pediatric tumors originating from the neural crest will be screened for the presence of polysialic acid. The size of the oligomers will be determined by ion exchange chromatography after brief treatment with Endo N. The genetic probes to polyalpha2- >8NeuNActransferase will be also used to screen the tumors. It is possible that variation in the length of sialyl polymers as seen in the conversion of the embryonic to adult forms of N-CAM, and/or the expression of the enzyme will aid in defining the pathology of these tumors.